Discovery and structure-activity relationships study of positive allosteric modulators of the M3 muscarinic acetylcholine receptor.

The M3 muscarinic acetylcholine receptor (mAChR) is a member of the family of mAChRs, which are associated with a variety of physiological functions including the contraction of various smooth muscle tissues, stimulation of glandular secretion, and regulation of a range of cholinergic processes in the central nerve system. We report here the discovery and a comprehensive structure--activity relationships (SARs) study of novel positive allosteric modulators (PAMs) of the M3 mAChR through a high throughput screening (HTS) campaign. Compound 9 exhibited potent in vitro PAM activity towards the M3 mAChR and significant enhancement of muscle contraction in a concentration-dependent manner when applied to isolated smooth muscle strips of rat bladder. Compound 9 also showed excellent subtype selectivity over other subtypes of mAChRs including M1, M2, and M4 mAChRs, and moderate selectivity over the M5 mAChR, indicating that compound 9 is an M3-preferring M3/M5 dual PAM. Moreover, compound 9 displayed acceptable pharmacokinetics profiles after oral dosing to rats. These results suggest that compound 9 may be a promising chemical probe for the M3 mAChR for further investigation of its pharmacological function both in vitro and in vivo.

New index of pain triggered by spinal activation of voltage-dependent sodium channels.

Voltage-dependent sodium channels (VDSCs) are crucial for pain generation. Here, to develop a new behavioral index of pain induced by spinal VDSC activation, we examined whether intrathecal veratridine injection produced nociceptive behavior. Intrathecal injection of the VDSC opener veratridine in mice dose-dependently induced nociceptive responses, with response times subsequently reduced by administration of morphine or pregabalin. Systemic administration of lidocaine and mexiletine, but not amitriptyline, also decreased this response time. Taken together, these results demonstrated that response time of nociceptive behavior induced by intrathecal veratridine injection is a quantitative index of pain triggered by spinal VDSC activation.

Spinal mechanism of standard analgesics: evaluation using mouse models of allodynia.

Spinal neurotransmission plays an important role in the perception of pain signaling. In the present study, we investigated the spinal anti-nociceptive mechanism of current standard analgesics in mouse models of tactile allodynia induced by intrathecal administration of N-methyl-D-aspartic acid (NMDA), prostaglandin E2 (PGE2), and bicuculline. NMDA-induced allodynia is induced by postsynaptic NMDA receptor activation, while PGE2-induced allodynia is triggered by the enhancement of presynaptic glutamate release via EP1 receptor activation. In contrast, bicuculline induces allodynia by the blockade of gamma-aminobutyric acid (GABA)A receptor-mediated inhibitory system. As the clinically available analgesics, pregabalin (alpha2delta-subunit calcium channel ligand), ziconotide (N-type calcium channel blocker), mexiletine (sodium channel blocker), and duloxetine (serotonin and norepinephrine reuptake inhibitors) were evaluated in these neurochemically-induced allodynia models. Pregabalin almost completely alleviated NMDA-, PGE2-, and bicuculline-induced allodynia. Despite being classified as an agent with a similar molecular target mechanism, ziconotide could only alleviate PGE2-induced allodynia, but not NMDA- or bicuculline-induced allodynia, as did mexiletine and duloxetine. These results taken together suggest that ziconotide, mexiletine, and duloxetine suppress spinal hyperactivity via the presynaptic site mechanism. In contrast, pregabalin could suppress via the downstream step during spinal hyperactivation such as postsynaptic NMDA activation or dysfunction of GABAergic control in addition to presynaptic mechanism. In conclusion, present findings provide implication that the spinal anti-nociceptive mechanistic site of pregabalin is different from that of ziconotide, mexiletine, and duloxetine, and pregabalin could have a broader anti-nociceptive mechanism other than N-type calcium channel blockade.

The involvement of aldose reductase in alterations to neurotrophin receptors and neuronal cytoskeletal protein mRNA levels in the dorsal root ganglion of streptozotocin-induced diabetic rats.

Dorsal root ganglia (DRG) are recognized as one of the organs which are damaged in peripheral sensory diabetic neuropathy. In an experimental animal model, the alteration of the mRNA expression level of neurotrophins, their receptors and neuronal cytoskeletal protein have been reported. In this study, we examined whether these changes are improved by treatment with the aldose reductase inhibitor, zenarestat, in early-stage diabetic neuropathy of streptozotocin (STZ)-induced diabetic rats. Two weeks after the induction of diabetes mellitus by STZ treatment, zenarestat or a vehicle were given orally for two weeks. After the zenarestat treatment, the mRNA expression levels of neurotrophin receptors and neuronal cytoskeletal proteins in dorsal root ganglia were determined with a real-time polymerase chain reaction (PCR) method. Compared with the expression level of normal rats, a significant increase in Trk-C and Talpha1 alpha-tubulin and a decrease in neurofilament H mRNA expression level were observed in the DRG of STZ rats, while there were no significant changes in Trk-A, Trk-B, p75, neurofilament L, neurofilament M and betaIII tubulin mRNA expression. Zenarestat treatment significantly ameliorated the abnormal increase in Trk-C mRNA expression level. These data suggest that hyperactivation of the polyol pathway induces a deficit in neurotropism on peripheral sensory diabetic neuropathy.

Pharmacological characterization of standard analgesics on mechanical allodynia in streptozotocin-induced diabetic rats.

The present study was designed to investigate the anti-allodynic effects of current analgesic agents, such as pregabalin, amitriptyline, mexiletine, morphine, and diclofenac, in a rat model of streptozotocin (STZ)-induced diabetic neuropathy. Diabetic rats developed a sustained decrease in withdrawal threshold response to the von Frey test within 8 weeks after a single injection of STZ (45 mg/kg, i.v.). The anti-allodynic effects of analgesic agents were examined after a single oral or subcutaneous administration at 3 and 7 weeks after beginning of STZ-treatment. Pregabalin (3-30 mg/kg, p.o.), an antiepileptic agent, dose-dependently blocked the mechanical allodynia in rats treated both at 3 and 7 weeks. Mexiletine (10-100 mg/kg, p.o.), a sodium channel blocker, dose-dependently ameliorated mechanical allodynia in rats treated at 3 weeks; however, the efficacy was diminished at 7 weeks. Morphine (1-10 mg/kg, s.c.) was effective in rats treated at 3 weeks; however, it was ineffective at 7 weeks. Conversely, an antidepressant amitriptyline (0.3-3 mg/kg, p.o.) improved mechanical allodynia in rats treated at 7 weeks, whereas it was ineffective at 3 weeks. Diclofenac, a non-steroidal anti-inflammatory drug, was ineffective at both time points. These results demonstrate that, except for diclofenac, the standard analgesic agents tested can effectively alleviate the mechanical allodynia seen in STZ-induced diabetic neuropathy. Their efficacies varied depending on the duration of the diabetic condition, suggesting that temporal changes in pharmacodynamic factors could affect the responsiveness of this model to analgesic agents.

Thirteen-month inhibition of aldose reductase by zenarestat prevents morphological abnormalities in the dorsal root ganglia of streptozotocin-induced diabetic rats.

The dorsal root ganglia (DRG) have been identified as the target tissue in diabetic somatosensory neuropathy. It has been reported that, in the chronically diabetic state, DRG sensory neurons may undergo morphological changes. In this study, we examined the effect of zenarestat, an aldose reductase inhibitor, on the morphological derangement of the DRG and the sural nerve of streptozotocin-induced diabetic rats (STZ rats) over a 13-month period. The cell area of the DRG in STZ rats was smaller than that in normal rats. A decrease in fiber size was apparent in the sural nerve of the STZ rats, and the fiber density was greater. These morphological changes were reversed in zenarestat-treated STZ rats. The data suggest that, in peripheral sensory diabetic neuropathy, hyperactivation of the polyol pathway induces abnormalities not only in peripheral nerve fiber, but also in the DRG, which is an aggregate of primary sensory afferent cell bodies.